The transcription factor NF-kB plays a pivotal role in many physiological and pathological processes, including immune and inflammatory responses. The activity of NF-kB is tightly regulated, primarily through phosphorylation and degradation of its inhibitors, IkB. The degradation of IkB by the ubiquitin- proteasome pathway allows NF-kB to enter the nucleus to regulate gene expression. Interestingly, phosphorylation of IkB by the kinase IKK is regulated by K63-linked polyubiquitination through a proteasome-independent mechanism. Our previous studies have shown that IKK activation requires the ubiquitin-conjugating enzyme complex consisting of Ubc13 and Uev1A, the ubiquitin ligase TRAF6, and the upstream protein kinase TAK1 complex. The TAK1 complex contains the adaptor proteins TAB2 and TAB3, which harbor specialized ubiquitin-binding domains capable of binding to K63 polyubiquitin chains. The binding of polyubiquitin chains to the TAK1 complex leads to the activation of this kinase, which in turn phosphorylates and activates IKK. IKK activation requires its regulatory subunit NEMO, which contains two ubiquitin-binding domains. The role of NEMO and its ubiquitin-binding in IKK activation is not fully understood. Our recent studies demonstrate that K63 polyubiquitination is essential for IKK activation by interleukin- 1? (IL-1?), which activates TRAF6. Moreover, we have found that TRAF6 and Ubc13/Uev1A catalyze the synthesis of unanchored polyubiquitin chains, which directly activate the TAK1 kinase complex. Interestingly, IKK activation by tumor necrosis factor-? (TNF?) requires polyubiquitination of RIP1 by Ubc5 and cIAPs as the E2 and E3, respectively, but does not require the formation of K63 polyubiquitin chains. These results suggest an unanticipated complexity of protein kinase regulation by ubiquitin. In this application, we propose to elucidate the distinct ubiquitin-dependent mechanisms of TAK1 and IKK activation in the IL-1? (Aim 1) and TNF? (Aim 2) pathways, respectively. In addition, we will investigate the mechanisms by which polyubiquitin binding by NEMO regulates IKK activation (Aim 3).